The invention relates to the field of biotechnology generally, and more specifically to adenoviral-based complementing cell lines.
Typically, vector and packaging cells have to be adapted to one another so that they have all the necessary elements, but they do not have overlapping elements which lead to replication competent virus by recombination. Therefore, the sequences necessary for proper transcription of the packaging construct may be heterologous regulatory sequences derived from, for example, other human adenovirus (Ad) serotypes, non-human adenoviruses, other viruses like, but not limited to, SV40, hepatitis B virus (HBV), Rous Sarcoma Virus (RSV), cytomegalo virus (CMV), etc. or from higher eukaryotes such as mammals. In general, these sequences include a promoter, enhancer and polyadenylation sequences.
PER.C6 (ECACC deposit number 96022940) is an example of a cell line devoid of sequence overlap between the packaging construct and the adenoviral vector (Fallaux et al., 1998). Recombinant viruses based on subgroup C adenoviruses such as Ad5 and Ad2 can be propagated efficiently on these packaging cells. Generation and propagation of adenoviruses from other serotypes, like subgroup B viruses, has proven to be more difficult on PER.C6 cells. However, as described in European patent application 00201738.2, recombinant viruses based on subgroup B virus Ad35 can be made by co-transfection of an expression construct containing the Ad35 early region-1 sequences (Ad35-E1). Furthermore, Ad35-based viruses that are deleted for E1A sequences were shown to replicate efficiently on PER.C6 cells. Thus, the E1A proteins of Ad5 complement Ad35-E1A functions, whereas at least part of the E1B functions of Ad35 are necessary. This serotype specificity in E1B functions was recently also described for Ad7 recombinant viruses. In an attempt to generate recombinant adenoviruses derived from subgroup B virus Ad7, Abrahamsen et al. (1997) were not able to generate E1-deleted viruses on 293 cells without contamination of wild-type (wt) Ad7. Viruses that were picked after plaque purification on 293-ORF6 cells (Brough et al., 1996) were shown to have incorporated Ad7 E1B sequences by non-homologous recombination. Thus, efficient propagation of Ad7 recombinant viruses proved possible only in the presence of Ad7-E1B expression and Ad5-E4-ORF6 expression. The E1B proteins are known to interact with cellular as well as viral proteins (Bridge et al., 1993; White, 1995). Possibly, the complex formed between the E1B 55K protein and E4-ORF6 which is necessary to increase MRNA export of viral proteins and to inhibit export of most cellular mRNAs, is critical and in some way serotype specific.
The present invention provides new packaging cell lines capable of complementing recombinant adenoviruses based on serotypes other than subgroup C viruses, such as serotypes from subgroup B, like adenovirus type 35.
In one aspect of the invention, the new packaging cells are derived from primary, diploid human cells such as, but not limited to, primary human retinoblasts, primary human embryonic kidney cells or primary human amniocytes. Transfection of primary cells with the adenovirus E1A gene alone can induce unlimited proliferation (immortalisation), but does not result in complete transformation. However, expression of E1A in most cases results in induction of programmed cell death (apoptosis), and occasionally immortalisation is obtained (Jochemsen et al., 1987). Co-expression of the E1B gene is required to prevent induction of apoptosis and for complete morphological transformation to occur (reviewed in White, 1995). Therefore, in one aspect of the invention, primary human cells are transformed by expression of adenovirus E1 proteins of a subgroup other than subgroup C, preferably subgroup B, more preferably adenovirus type 35. The combined activity of the E1A and E1B proteins establishes indefinite growth of the cells and enables complementation of recombinant adenoviruses.
In another aspect of the invention, the transforming E1 sequences are derived from different serotypes. As disclosed in European Patent application 00201738.2, Ad35 E1 sequences are capable of transforming Baby Rat Kidney (BRK) cells, albeit with a lower efficiency than that seen with Ad5 E1 sequences. This was also observed for E1 sequences from Ad12 (Bernards et al., 1982). Therefore, in this aspect of the invention, primary diploid human cells are transformed with chimeric E1 constructs that consist of part of the E1 sequences of a serotype that enables efficient transformation of primary human cells and part of the E1 sequences of another serotype which E1 sequences provide the serotype-specific E1B function(s) that enable(s) efficient propagation of E1-deleted viruses of that serotype. In a preferred embodiment of this aspect of the invention, the E1A region is derived from a subgroup C adenovirus, like, but not limited to, Ad5, and the E1B sequences are derived from an alternative adenovirus more particularly from an adenovirus of subgroup B, more particularly from adenovirus type 35. In a more preferred embodiment, the E1A sequences and the E1B-21K sequences are derived from a subgroup C adenovirus, like, but not limited to, Ad5, and the E1B-55k sequences as far as not overlapping with the 21K sequences are derived from an adenovirus of subgroup B, more particular from adenovirus type 35. In an even more preferred embodiment, all E1 sequences are derived from a subgroup C adenovirus, like but not limited to Ad5, except for at least the part of the E1B-55K sequences that are necessary for serotype-specific complementation of an alternative adenovirus subgroup, more particular adenovirus subgroup B, more particular adenovirus type 35, the sequences being derived from the adenovirus.
The primary diploid human cells are transformed by adenovirus E1 sequences either operatively linked on one DNA molecule or located on two separate DNA molecules. In the latter case, one DNA molecule carries at least part of the E1 sequences of the serotype enabling efficient transformation and the second DNA molecule carries at least part of the sequences necessary for serotype-specific complementation. In all aspects, the sequences are operatively linked to regulatory sequences enabling transcription and translation of the encoded proteins.
In another aspect of the invention, new packaging cells are described that are derived from PER.C6 (ECACC deposit number 96022940; Fallaux et al., 1998) and contain Ad35-E1 sequences integrated into their genome. These Ad35-E1 sequences are present in a functional expression cassette, but preferably do not contain sequences overlapping with sequences present in the recombinant viral vector. Preferably, the functional expression cassette consists of a heterologous promoter and poly-adenylation signal functionally linked to Ad35-E1 sequences. More specifically, the Ad35-E1 sequences are functionally linked to the human phosphoglycerate gene promoter (hPGK) and hepatitis B virus poly-adenylation signal (HBV-pA). Preferably, Ad35-E1 sequences comprise the coding regions of the E1A proteins and the E1B promoter sequences linked to E1B coding sequences up to and including the stop codon of the E1B 55K protein. More preferably, the Ad35-E1 sequences comprise nucleotide 468 to nucleotide 3400 of the Ad35 wt sequence. To be able to select for transfected cells, a dominant selection marker like, but not limited to, the neor gene has to be incorporated on the expression vector or the Ad35 expression vector is co-transfected with a separate expression vector mediating expression of the selection marker. In both cases, the selection marker becomes integrated in the cellular genome. Other AdS-E1 transformed cell lines like 293 (Graham et al., 1977) and 911 (Fallaux et al., 1996) or established human cell lines like A549 cells may be used without departing from the present invention.
In another aspect of the invention, PER.C6-derived cells are described that express functional Ad35 E1B sequences. In one embodiment, the Ad35-E1B sequences are driven by the E1B promoter and terminated by a heterologous poly-adenylation signal like, but not limited to, the HBVpA. In a preferred embodiment, the Ad35-E1B sequences are driven by a heterologous promoter like, but not limited to, the hPGK promoter or Elongation Factor-1xcex1 (EF-1xcex1) promoter and terminated by a heterologous pA signal like, but not limited to, the HBVpA. These Ad35-E1B sequences preferably comprise the coding regions of the E1B 21K and the E1B 55K proteins located between nucleotides 1611 and 3400 of the wild-type (wt) Ad35 sequence. More preferably, the Ad35-E1B sequences comprise nucleotides 1550 to 3400 of the wt Ad35 sequence. In an even more preferred embodiment, the E1B sequences comprise the coding sequences of the E1B-55K gene located between nucleotides 1916 and 3400 of the wt Ad35 sequence.
Cell lines subject of this invention are useful for, among other things, the production of recombinant adenoviruses designed for gene therapy and vaccination. The cell lines, being derived from cells of human origin, are also useful for the production of human recombinant therapeutic proteins like, but not limited to human growth factors, human antibodies. In addition the cell lines are useful for the production of human viruses other than adenovirus like, but not limited to, influenza virus, herpes simplex virus, rotavirus, measles virus.